The regulatory mechanisms for cellular proteolysis under varied nutritional conditions remain poorly understood, although a balance between protein synthesis and degradation would appear to be a fundamental requirement of living cells to provide homeostasis throughout the cell cycle. Numerous studies of the lysosomal and nonlysosomal proteolysis of a variety of proteins have produced conflicting results. We propose to investigate cellular proteolysis by taking advantage of the unique characteristic of the human mutation, cystinosis, which results in the inability of these cells to transport cystine out of the lysosome once it has been formed there from the degradation of cystine-containing proteins. We will prepare purified proteins metabolically labelled with 35S-cystine in their disulfide moieties and study the resultant lysosomal 35S-cystine accumulation in cystinotic fibroblasts produced after introducing them to the cell via endocytosis, pinocytosis or microinjection. The results should enhance our understanding of the loci and regulatory mechanisms of cellular proteolysis.